The present invention, in some embodiments thereof, relates to the use of agents which regulate alternative splicing pathways for the treatment and diagnosis of cancer and related diseases.
The process of alternative splicing is widely misregulated in cancer and many tumors express new splicing isoforms, which are absent in the corresponding normal tissue. Many oncogenes and tumor suppressors are differentially spliced in cancer cells and it has been shown that many of these cancer-specific isoforms contribute to the transformed phenotype of cancer.
Splicing factor SRSF1 (SF2/ASF) is a potent proto-oncogene. It is upregulated by several mechanisms such as gene amplification (Karni et al. 2007) or transcriptional activation (Das et al. 2012). SRSF1 modulates the splicing of numerous genes including Mnk2, RPS6KB1 which encodes S6K1 in both mouse and human.
The serine/threonine kinases Mnk1 and Mnk2 were discovered by their direct interaction with and activation by the MAP kinases ERK and p38 Mnk1 and Mnk2 phosphorylate the translation initiation factor eIF4E on serine 209. The eIF4E protein binds to the 5′ cap structure of mRNAs and is essential for cap-dependent translational initiation. In mice lacking both kinases (MNK-DKO mice) eIF4E is completely unphosphorylated on serine 209. Intriguingly, these mice develop and live normally displaying no adverse phenotype. Mnk1 and Mnk2 are 72% identical in their amino acid sequence. Biochemically, it has been shown that while Mnk1 is activated only after stimulation of the upstream MAPK signaling, Mnk2 possesses intrinsic basal activity when introduced into cells. There is no direct evidence connecting Mnk1/2 to human cancer. It would seem that the notion that Mnk1 and Mnk2 are positive drivers in human cancer stems from the important role eIF4E, their known substrate, plays in cancer.
In humans, each of the MKNK1 and MKNK2 genes gives rise to at least two distinct proteins, with different C termini, as a consequence of 3′ prime alternative splicing. The longer forms of human Mnk1 and Mnk2, referred to as Mnk1a and Mnk2a respectively, possess a MAPK-binding motif that is absent from the shorter isoforms Mnk1b and Mnk2b.
Adesso et al. 2012, [Oncogene, doi: 10.1038/onc.2012.306.] teach that resistance of pancreatic cancer cells to Gemcitabine is mediated by SRSF1 up-regulation and a switch in Mnk2 alternative splicing, which enhances eIF4E phosphorylation implicating this alternative splicing event with chemotherapy resistance.
The PI3K/Akt/mTOR pathway is one of the major signaling pathways hyper activated in many cancers, and leads to uncontrolled proliferation, increased survival, motility and invasiveness of cancer cells. mTOR resides in two distinct complexes: mTOR complex-1 (mTORC1) and complex-2 (mTORC2). mTORC1 core contains mTOR, Raptor, G-β-L and is considered to be sensitive to rapamycin. mTORC2 contains Rictor, as the mTOR partner instead of Raptor, and depending on the cell type, is less sensitive to rapamycin. The best-characterized substrates of mTORC1 are S6 Kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), while Akt is a substrate of mTORC2. Several components of the mTOR signaling cascade have been identified as oncogenes or tumor suppressors that activate or repress this pathway respectively. Among the two well characterized mTORC1 substrates, S6K1 and 4E-BP1, the latter has been shown to be important for efficient protein translation, proliferation, and for oncogenic transformation. S6K1 has been implicated in the regulation of cell size. A link between S6K1 function and cancer was suggested by the finding that RPS6KB1, the gene encoding for S6K1, resides in the chromosomal region 17q22-17q23, which is often amplified in breast and lung cancers (Bepler and Koehler, 1995; Monni et al., 2001). However, direct evidence that S6K1 expression or activity is sufficient to lead to cellular transformation is lacking.
Karni et al. 2007, [Nature structural & molecular biology 14: 185-193] teach that SRSF1 increases the expression of the shorter S6K1 isoform (referred to herein as h6A and h6C in human) as opposed to the long active kinase p85/p70 S6K1 (referred to herein as Iso-1).